The long term goals of this project are to gain an understanding of the functional importance and development of the high affinity (HA) uptake systems for catecholamines and serotonin in primary astrocyte cultures, as well as an alpha-receptor induced depolarization, to help elucidate the roles and functions of astroglia in situ. We propose to see whether variation in the expression of these uptake systems are receptor affects occur in astrocyte cultures prepared from a diversity of brain regions. We will prepare immunocytochemically defined astrocyte cultures from different regions of 1 day old rat brain. We will study HA uptake of the catecholamines and serotonin, and further analyze the mechanisms of action of the norepinephrines induced depoloarization in terms of ionic mechanisms and pharmacology. We will also examine the beta-receptor stimulated cAMP response in different cultures. We will study the activities of the different metabolizing enzymes for these transmitters, monoamine oxidase (MAO) and catechol-O-methyl transferase (COMT), as well as the production of metabolites. To see if HA uptake systems are present in astrocytes in situ, we will study uptake of (3H) labelled serotonin and also double label for glial fibrillary acidic protein in tissue sections from different regions of 20-40 day old rat brain and also study such uptake by electron microscope autoradiography. The existence of such HA uptake and receptors for monoamines is neuronal-free astrocyte cultures suggests that the expression of these systems is an innate developmental property of these cells which does not require continuous neuronal or other external cues. To try to establish when this occurs we will study cultures prepared from 15 day fetal through 6 to 10 day old rats. These cultures will be analyzed immunocytochemically and we will take care to remove any contaminating neurons or other cells. In order to determine possible neuronal influences on established cultures we will co-culture astrocytes with neuronal cultures prepared from the mesencephalon and the pons + brains stem of fetal rats and study uptake by autoradiography. These studies should enable us to draw conclusions regarding several important aspects of HA monoamine uptake and receptors on astrocytes; namely influences on the development of such systems, whether the astrocytic HA uptake system exists in situ and how the activity of such systems compares to the activity of neurons. These studies have implications for the role of astrocytes in brain function, psychiatric disorders such as depression and possibly recent finding on toxin (MPTP) induced Parkinsonism.